JP7703541B2 - Serum albumin-binding fibronectin type III domain and uses thereof - Google Patents

Description

The present embodiments relate to fibronectin type III (FN3) domains that bind serum albumin. Such FN3 domains can be used, for example, to extend the in vivo serum half-life of drugs or proteins conjugated thereto. Also provided are methods for producing such molecules and pharmaceutical compositions containing them.

Rapid clearance of drugs or biotherapeutic molecules from the bloodstream may contribute to limited clinical effectiveness of these molecules or result in more frequent dosing for patients. One commonly occurring method of clearance is renal clearance by glomerular filtration. This route of clearance is most relevant for smaller biotherapeutics, as renal filtration rates are significantly reduced for molecules with molecular weights above 50,000 daltons (Non-Patent Document 1). Several approved biotherapeutics contain active moieties that are themselves below the filtration limit and therefore rapidly cleared. To overcome this limitation, a number of techniques have been introduced to effectively increase the size of therapeutic molecules, thus reducing renal filtration.

Kontermann, Curr Opin Biotechnol 2011

There remains a need for compounds or methods for extending the half-life of drugs or therapeutic agents. The present embodiments meet these needs, as well as others.

The present embodiments provide serum albumin-binding fibronectin type III (FN3) domains. Also described are associated polynucleotides capable of encoding the provided FN3 domains, cells expressing the provided FN3 domains, as well as associated vectors. Additionally, methods of using the provided FN3 domains are described. For example, given the long serum half-life of albumin, albumin-binding peptides can be used as fusion partners to create therapeutic proteins or drugs with long or extended serum half-lives.

In addition to the albumin-binding FN3 domains, polynucleotide sequences capable of encoding the described proteins are also provided. Vectors comprising the described polynucleotides are also provided, as are cells expressing the albumin-binding FN3 domains of the present specification. Cells capable of expressing the disclosed vectors are also described. These cells can be mammalian cells (such as 293F cells, CHO cells, etc.), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as E. coli). Methods for producing the described FN3 domains (proteins) are also provided.

The present embodiments also provide methods for fusing or otherwise associating the provided albumin-binding FN3 domains to various molecules to extend the half-life of such molecules. In this manner, the albumin-binding FN3 domains can be used, for example, to extend the half-life of therapeutic agents.

In some embodiments, pharmaceutical compositions comprising an albumin-binding FN3 domain are administered to improve the in vivo half-life of the therapeutic partner. Such half-life extension can be assayed by a variety of methods known in the art, including, for example, monitoring the pharmacokinetics of the albumin-binding FN3 domain. Non-limiting examples of such assays for these methods are provided in the Examples provided herein.

Kits are also provided that include the albumin-binding FN3 domains provided. The kits can be used to carry out methods using the albumin-binding FN3 domains provided herein, or other methods known to those of skill in the art. In some embodiments, the described kits may include the FN3 domains described herein and reagents for use in detecting the presence of human serum albumin in a biological sample. The described kits may include one or more of the FN3 domains described herein, as well as a container for housing the FN3 domain when not in use, instructions for use of the FN3 domain immobilized on a solid support, and/or a detectably labeled form of the FN3 domain, as described herein.

FIG. 1 shows the indirect pull-down of endogenous albumin from cynomolgus monkey and human serum using albumin-binding FN3 domains. FN3 domains that bind to domain 1 of albumin are surrounded by dotted lines; FN3 domains that bind to domain 3 of albumin are surrounded by solid lines. All constructs are prepared as bispecific gene fusions with one null FN3 domain (TC25) and one albumin-binding FN3 domain. ^* ABD stands for albumin-binding domain (Reference: J.T.Andersen, R.Pehrson, V.Tolmachev, M.B.Daba, L.Abrahmsen, C.Ekblad, J.Biol.Chem.286:5234-5241,2011). FIG. 2 shows the pharmacokinetic profile of albumin-binding FN3 domains in cynomolgus monkeys following a single IV dose of 5 mg/kg (H9) or 5 mg/kg (ALB40(B7)).

Definitions Various terms relating to the described aspects are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

The term "about" as used herein when referring to measurable values, such as amounts, time durations, and the like, is meant to encompass variations of up to ±10% from the specified value, with such variations being appropriate for carrying out the disclosed method. Unless otherwise indicated, all numbers expressing properties such as amounts of ingredients, molecular weights, reaction conditions, and the like used in the specification and claims should be understood in all instances to be modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, such numerical values inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

"Isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced away, or purified away from other biological components of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition, and still be isolated if such a composition is not part of the natural environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, an "isolated" FN3 domain is intended to refer to an FN3 domain that is substantially free of other FN3 domains with different antigen specificities (e.g., an isolated FN3 domain that specifically binds human serum albumin is substantially free of FN3 domains that specifically bind antigens other than human serum albumin). However, an isolated FN3 domain that specifically binds to an epitope, isoform or variant of human serum albumin may have cross-reactivity to other related antigens, for example from other species (such as serum albumin species homologues).

As used herein, the term "fibronectin type III (FN3) domain" (FN3 domain) refers to a domain that occurs frequently in proteins including fibronectins, tenascins, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin-C, the 15 different FN3 domains present in human fibronectin (FN), and non-naturally occurring synthetic FN3 domains, e.g., as described in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the third FN3 domain of tenascin (TN3), or the tenth FN3 domain of fibronectin (FN10).

The term "specifically binds" or "specific binding" as used herein refers to the ability of the FN3 domains of the invention to bind to a given antigen with a dissociation constant (K D ) of about 1×10 ⁻⁶ M or less, e.g., about 1× ^{10 −7 M} or less, about 1×10 ⁻⁸ M or less, about 1×10 ⁻⁹ M or less, about 1×10 ⁻¹⁰ M or less, about 1×10 ⁻¹¹ M or less, about 1×10 −12 M or less, or about 1×10 ⁻¹³ M or less. Typically, the described FN3 domains bind to a given antigen (i.e., human serum albumin) with a K _D that is at least 10-fold less than its K _D for a non-specific antigen (e.g., casein) as measured, for example, by surface plasmon _resonance using a Proteon Instrument (BioRad). However, the described FN3 domains that specifically bind human serum albumin may have cross-reactivity to other related antigens, e.g., the same given antigen from other species (homologs), such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

As used herein, "serum half-life" may generally be defined as the time required for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, e.g., due to degradation of the sequence or compound and/or clearance or sequestration by natural mechanisms of the sequence or compound. The in vivo half-life of the amino acid sequences, compounds, or polypeptides of the invention may be determined by any method known per se, e.g., pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally comprise the steps of administering a suitable dose of an amino acid sequence, compound or polypeptide of the present disclosure to a warm-blooded animal (i.e., a human or another suitable mammal such as a mouse, rabbit, rat, pig, dog, primate, for example monkeys of the genus Macaca (particularly Cynomolgus monkeys (Macaca fascicularis) and/or Rhesus monkeys (Macaca mulatta) and baboons (Papio ursinus)); collecting a blood or other sample from said animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the present invention in said blood sample; and calculating from (a plot of) the data so obtained the time until the level or concentration of the amino acid sequence, compound or polypeptide of the present invention is reduced by 50% upon administration compared to the initial level. See, for example, the experimental part below, as well as standard handbooks such as Kenneth, A. et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996). See also "Pharmacokinetics", M. Gibaldi and D. Perron, Marcel Dekker Publishing, revised 2nd edition (1982).

As will also be appreciated by those skilled in the art (see, for example, pages 6 and 7 of WO 04/003019 and further references cited therein), half-life can be expressed in terms of parameters such as t1/2-alpha, t1/2-beta and area under the curve (AUC). As used herein, "half-life" refers to the decrease in any one of these parameters, such as any two of these parameters, or essentially all three of these parameters.

The term "pharmacokinetics" or "pharmacokinetics" is used according to its art-recognized meaning to mean the study of the action of drugs in the body, including the efficacy and duration of action of drugs and the rates at which they are absorbed, distributed, metabolized, and eliminated by the body.

As used herein, the terms "substitution" or "substituted" or "mutation" or "mutated" refer to the alteration, deletion, or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to produce a variant of that sequence.

As used herein, the term "randomization" or "randomized" or "diversification" or "diversified" refers to at least one substitution, insertion or deletion in a polynucleotide or polypeptide sequence.

As used herein, "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as, for example, a substitution, insertion, or deletion.

The term "library" refers to a collection of variants. A library is composed of variants of polypeptides or polynucleotides.

As used herein, "Tencon" refers to a synthetic fibronectin type III (FN3) domain having the sequence set forth in SEQ ID NO:1 and described in U.S. Patent Application Publication No. 2010/0216708.

"Polynucleotide", referred to interchangeably as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to triple-stranded regions that include RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases for stability or other reasons, and DNA or RNA with modified backbones. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides as they are commonly found in nature, as well as the chemical forms of DNA and RNA that are characteristic of viruses and cells. "Polynucleotide" also includes relatively short strands of nucleic acid often called oligonucleotides.

A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment is operably inserted so as to bring about the replication or expression of the segment.

The term "host cell" as used herein can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In certain embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule, and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to possible mutations or environmental influences in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome. The terms "expression" and "production" are used interchangeably herein and refer to the biosynthesis of a gene product. These terms include transcription of a gene into RNA. These terms also include translation of RNA into one or more polypeptides, and further include all naturally occurring post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof may be within the cytoplasm of a cell, or within an extracellular environment, such as the growth medium of a cell culture. The meaning of "substantially identical" may vary depending on the context in which the term is used. Due to natural sequence variation likely to exist among heavy and light chains and the genes encoding them, it is expected that some variation will be found within the amino acid sequences or genes encoding the antibodies or antigen-binding fragments described herein that has little or no effect on the inherent binding properties (e.g., specificity and affinity). Such an expectation is due, in part, to the degeneracy of the genetic code and the evolutionary success of conservative amino acid sequence variations that do not appreciably alter the properties of the encoded proteins.

Overview of Disclosed FN3 Domains Tencon (SEQ ID NO:1) is a non-naturally occurring fibronectin type III (FN3) domain designed from the consensus sequence of 15 FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Patent Application Publication No. 2010/0216708). The crystal structure of Tencon shows six surface-exposed loops connecting seven beta strands characteristic of FN3 domains, the beta strands designated A, B, C, D, E, F, G, and the loops designated AB, BC, CD, DE, EF, FG loops (Bork and Doolittle, Proc Natl Acad Science USA 89:8990-8992, 1992; U.S. Patent No. 6,673,901). These loops, or selected residues within each loop, may be randomized to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind serum albumin. Table 1 shows the location and sequence of each loop and beta strand of Tencon (SEQ ID NO:1).

Thus, libraries designed based on Tencon sequences may have randomized FG loops, or randomized BC and FG loops, such as libraries TCL1 or TCL2 described below. The BC loop of Tencon is 7 amino acids long, and thus 1, 2, 3, 4, 5, 6, or 7 amino acids are diversified in the BC loop and randomized in libraries designed based on Tencon sequences. The FG loop of Tencon is 7 amino acids long, and thus 1, 2, 3, 4, 5, 6, or 7 amino acids are diversified in the FG loop and randomized in libraries designed based on Tencon sequences. Further diversity in the loops of the Tencon library may be achieved by insertion and/or deletion of residues in the loops. For example, the FG and/or BC loops may be extended by 1-22 amino acids or reduced by 1-3 amino acids. The Tencon FG loop is 7 amino acids long, while the corresponding loop in an antibody heavy chain ranges from 4 to 28 residues. To provide maximum diversity, the FG loop is diversified in sequence and length to correspond to the range of CDR3 lengths of antibodies, 4 to 28 residues. For example, the FG loop is further diversified in length by extending the loop with an additional 1, 2, 3, 4 or 5 amino acids.

Libraries designed based on Tencon sequences may also have randomized alternative surfaces formed on the sides of the FN3 domain and including two or more beta strands and at least one loop. One such alternative surface is formed by amino acids of the C and F beta strands and the CD and FG loops (C-CD-F-FG surface). Library designs based on Tencon alternative C-CD-F-FG surfaces are described in US Patent Application Publication No. 2013/0226834. Libraries designed based on Tencon sequences also include libraries designed based on Tencon variants, such as Tencon variants with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbers corresponding to SEQ ID NO:1) that exhibit improved thermostability. Exemplary Tencon variants are described in U.S. Patent Application Publication No. 2011/0274623 and include Tencon27 (SEQ ID NO:4), which has substitutions E11R, L17A, N46V, and E86I when compared to the Tencon of SEQ ID NO:1.

Tencon and other FN3 sequence-based libraries are randomized at selected residue positions using a set of random or defined amino acids. For example, variants in libraries with random substitutions can be generated using the NNK codon, which codes for all 20 naturally occurring amino acids. In other diversification schemes, the DVK codon can be used to code for the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, the NNS codon can be used to generate all 20 amino acid residues and simultaneously reduce the frequency of stop codons. Libraries of FN3 domains with biased amino acid distributions at diversified positions can be synthesized, for example, using Slonomics® technology (http://www_sloning_com). This technology uses a library of premade double-stranded triplets that serve as universal building blocks sufficient for thousands of gene synthesis processes. The library of triplets represents all possible sequence combinations necessary to construct any desired DNA molecule. Codon designations are according to the well-known IUB code.

The FN3 domains that bind or specifically bind human serum albumin described herein can be isolated by using cis display to link a DNA fragment encoding a scaffold protein to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation in which each protein is stably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), creating an FN3 library such as the Tencon library, and assaying the library for specific binding to human serum albumin by any method known in the art and described in the Examples. Exemplary well-known methods that can be used are ELISA, sandwich immunoassays, and competitive and noncompetitive assays (see, e.g., Current Protocols in Molecular Biology, Vol. 1, eds. Ausubel et al., 1994, John Wiley & Sons, Inc., New York). Identified FN3 domains that specifically bind to human serum albumin are further characterized according to desired properties.

The FN3 domains that specifically bind human serum albumin described herein can be generated by using any FN3 domain as a template to generate a library and screening the library for molecules that specifically bind human serum albumin using the methods provided. Exemplary FN3 domains that can be used are the third FN3 domain of tenascin-C (TN3) (SEQ ID NO: 81), fibcon (SEQ ID NO: 82), and the tenth FN3 domain of fibronectin (FN10) (SEQ ID NO: 83). To express or translate the library in vitro, standard cloning and expression techniques are used to clone the library into a vector or to synthesize a double-stranded cDNA cassette of the library. For example, ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA 94, 12297-12302, 1997), or other cell-free systems (U.S. Pat. No. 5,643,768) can be used. The library of FN3 domain variants is expressed, for example, as a fusion protein displayed on the surface of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of bacteriophage are well known (U.S. Patent Publication No. 2011/0118144; WO 2009/085462; U.S. Patent No. 6,969,108; U.S. Patent No. 6,172,197; U.S. Patent No. 5,223,409; U.S. Patent No. 6,582,915; U.S. Patent No. 6,472,147).

In some embodiments described herein, the FN3 domain that specifically binds human serum albumin is based on the Tencon sequence of SEQ ID NO:1 or the Tencon27 sequence of SEQ ID NO:4, optionally with substitutions at residue positions 11, 14, 17, 37, 46, 73, and/or 86 of SEQ ID NO:1 or SEQ ID NO:4.

The FN3 domains that specifically bind human serum albumin of the present disclosure are modified to improve their properties, such as improving thermal stability and reversibility of thermal folding and unfolding. To increase the apparent thermal stability of proteins and enzymes, several methods have been applied, including rational design based on comparison with highly similar thermostable sequences, stabilizing design of disulfide bridges, mutations that increase alpha-helical propensity, modification of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability can increase the yield of expressed proteins, improve solubility or activity, reduce immunogenicity, and minimize the need for a cold chain in production. Residues that can be substituted to improve the thermal stability of Tencon (SEQ ID NO:1) are at residue positions 11, 14, 17, 37, 46, 73, or 86 and are described in U.S. Patent Application Publication No. 2011/0274623. Substitutions corresponding to these residues can be incorporated into the FN3 domain containing molecules of the invention.

Measurements of protein stability and protein instability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or "unstable" to denaturation caused by heat, ultraviolet or ionizing radiation, changes in the surrounding osmolarity and pH when in solution, mechanical shear forces imposed by filtration through small pore sizes, ultraviolet radiation, ionizing radiation such as by gamma irradiation, chemical or thermal dehydration, or any other action or force that can cause the destruction of protein structure. Molecular stability can be determined using standard methods. For example, molecular stability can be determined using standard methods by measuring the thermal melting (" _Tm ") temperature, which is the temperature in degrees Celsius (°C) at which half of the molecule unfolds. Typically, the higher the _Tm , the more stable the molecule. In addition to heat, the chemical environment also changes the ability of a protein to maintain a specific three-dimensional structure.

In one embodiment, an FN3 domain that specifically binds human _serum albumin of the present disclosure exhibits increased stability of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same domain prior to modification as measured by an increase in Tm.

Chemical denaturation can likewise be measured by a variety of methods. Chemical denaturants include guanidine hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride), reducing agents (e.g., hydrides such as dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and sodium borohydride), non-ionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH ₃ COOH), acetic acid halides), hydrophobic molecules (e.g., phospholipids), and targeted denaturants. Quantification of the extent of denaturation can be based on loss of functional properties, such as the ability to bind target molecules, or on physicochemical properties, such as the tendency to aggregate, exposure of previously solvent inaccessible residues, or the breaking or formation of disulfide bonds.

The FN3 domains of the present disclosure can be generated as monomers, dimers, or multimers, for example, as a means of increasing the valency and thus the avidity of target molecule binding, or as a means of generating bi- or multi-specific scaffolds that bind two or more different target molecules simultaneously. Dimers and multimers can be generated, for example, by linking monospecific, bi- or multispecific protein scaffolds by including an amino acid linker, for example, a linker containing polyglycine, glycine and serine, or alanine and proline. Exemplary linkers include (GS) ₂ (SEQ ID NO: 71), (GGGS) 2 (SEQ ID NO: 72), (GGGGS) ₅ (SEQ ID NO: 73), (AP) ₂ (SEQ ID NO: 74), (AP) ₅ (SEQ ID NO: ₇₅ ), (AP) ₁₀ (SEQ ID NO: 76), (AP) ₂₀ (SEQ ID NO: 77), and A(EAAAK) ₅ AAA (SEQ ID NO: 78). The dimers and multimers are linked to each other in the N to C direction. The use of naturally occurring and artificial peptide linkers to connect polypeptides into novel ligated fusion polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson and Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456).

Human serum albumin binders Due to their small size of about 10 kDa, FN3 domains are rapidly removed from the circulation by renal filtration and degradation. In certain aspects, the present disclosure provides FN3 domains that specifically bind to serum albumin, e.g., human serum albumin (HSA), to extend the half-life of the FN3 domain or another therapeutic agent to which the albumin-binding FN3 domain is associated or linked.

In some embodiments, the human serum albumin-binding FN3 domain comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the human serum albumin-binding FN3 domain comprises a cysteine (Cys) linked to the C-terminus or N-terminus of the FN3 domain.

The addition of an N-terminal Met and/or a C-terminal Cys may facilitate expression and/or conjugation to another molecule, which may be another half-life extending molecule, such as PEG, an Fc region, another FN3 domain, etc.

In some embodiments, the FN3 domain comprises the amino acid sequence of SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In some embodiments, the FN3 domain (protein) is isolated. In some embodiments, the FN3 protein comprises SEQ ID NO:51 with at least one substitution compared to SEQ ID NO:51. In some embodiments, the substitution is at the residue corresponding to position 10 of SEQ ID NO:51. In some embodiments, the substitution (mutation) is A10V. In some embodiments, the substitution is A10 to G, L, I, T, or S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid.

In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence of SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In some embodiments, the FN3 domain is selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, provided that the protein has a substitution corresponding to position 10 of the amino acid sequence of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. 68, or 69. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid.

In some embodiments, the isolated FN3 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 substitutions compared to the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In some embodiments, the substitutions are at positions corresponding to position 10 of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69.

In some embodiments, the provided FN3 domains include a cysteine residue at at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88, or positions 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, or 93 of SEQ ID NO:1, or at the C-terminus. Although a series of positions are listed, each position can also be selected individually. In some embodiments, the cysteine is at a position corresponding to position 6, position 53, or position 88.

In certain embodiments, the isolated FN3 domains described herein have C strands, CD loops, F strands, and FG loops that are identical to those of the described albumin-binding FN3 domain sequences (i.e., SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69) or the C strands, CD loops, F strands, and FG loops of the four core FN3 domain sequences. The sequence may include a sequence set forth in SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, in which the C strand, CD loop, F strand, and FG loop are replaced with a respective set of C strand, CD loop, F strand, and FG loop from any of a sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence of the FG loop.

In some embodiments, the isolated albumin-binding FN3 domain comprises a sequence set forth in SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69.

In some embodiments, the FN3 domain comprises SEQ ID NO:51 with a substitution at position 10, 33, 25, 37, 39, 40, 41, 42, 47, 49, 69, 71, 73, 79, 80, 82, 85, or 87. In some embodiments, the FN3 domain comprises SEQ ID NO:51 with a substitution at position 10. In some embodiments, the FN3 domain comprises SEQ ID NO:51 with a substitution at position 10 and an additional substitution of A10V, A10G, A10L, A10I, A10T, or A10S.

In some embodiments, the FN3 domain comprises an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:51 or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions when compared to the amino acid sequence of SEQ ID NO:51.

In some embodiments, the substitution is an alanine substitution. In some embodiments, the substitution is G, L, I, T, or S. In some embodiments, the substitution or change is to any other naturally occurring amino acid residue. "Naturally occurring amino acid residue" refers to the 20 amino acid residues, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, W37 or W47 is substituted with a different hydrophobic residue, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, or methionine. In some embodiments, W37 or W47 is substituted with phenylalanine or tyrosine.

Fusion of a human serum albumin-binding FN3 domain In some embodiments, the present disclosure provides a conjugate comprising a serum albumin-binding FN3 domain and at least one additional moiety. The additional moiety may be useful for any diagnostic, imaging, or therapeutic purpose. In some embodiments, the additional moiety is an antisense oligonucleotide, an siRNA, an miRNA, an antibody, another FN3 domain, etc. Other examples of FN3 domains include, but are not limited to, those provided in U.S. Pat. Nos. 8,278,419, 9,200,059, 10,040,842, 8,569,227, 9,234,029, 9,982,253, 9,200,273, 9,897,612, 10,196,446, 8,415,291, 8,617,894, 9,695,228, 9,725,497, 9,156,887, or 10,280,200, each of which is incorporated by reference in its entirety with reference to the specific FN3 domains provided therein. and incorporated in their entirety, including the specific FN3 domains provided therein, or those provided in U.S. Patent Application Nos. 15/629090, 16/218990, 15/637276, 15/148312, 15/611296, 15/839915, 15/840281, 15/840303, 62/914643, 62/914654, or 62/914725, each of which is incorporated by reference in its entirety, including the specific FN3 domains provided therein.

In certain embodiments, the serum half-life of the moiety fused to the described FN3 domain is increased compared to the serum half-life of the moiety when not conjugated to the FN3 domain. In certain embodiments, the serum half-life of the FN3 domain fusion is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 1900, 2000, 2500, or 3000% longer than the serum half-life of the moiety when not fused to the described FN3 domain. In other embodiments, the serum half-life of the FN3 domain fusion is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40, or 50 times longer than the serum half-life of the moiety when not fused to the FN3 domain described. In some embodiments, the serum half-life of the FN3 domain fusion is at least 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, or 40 hours in cynomolgus monkeys.

Thus, the FN3 domain fusion molecules described herein are useful for increasing the half-life of a therapeutic moiety by creating a fusion between the therapeutic moiety and the described FN3 domain. Such fusion molecules can be used to treat conditions that are responsive to the biological activity of the therapeutic moiety contained in the fusion. The present disclosure contemplates the use of the described FN3 domain fusion molecules in diseases caused by dysregulation of any of the following proteins or molecules:

Heterologous Moieties In some embodiments, the described FN3 domains are fused to a second moiety that is a small organic molecule, a nucleic acid molecule, or a protein. In some embodiments, the described FN3 domains are fused to a therapeutic moiety that targets a receptor, a receptor ligand, a viral coat protein, an immune system protein, a hormone, an enzyme, an antigen, or a cell signaling protein. The fusion can be formed by attaching a second moiety to either end of the described FN3 domain, i.e., in the following configuration: FN3 domain-therapeutic molecule or therapeutic molecule-FN3 domain.

In other exemplary embodiments, the described FN3 domains are fused to one or more additional FN3 domains. For example, the described FN3 domains are fused to one, two, three, four, or more additional FN3 domains. The additional FN3 domains may bind to the same or different targets other than serum albumin. Examples of FN3 domains are provided herein and incorporated by reference.

In some embodiments, the described FN3 domain is linked to another molecule. In some embodiments, the other molecule is a drug or therapeutic agent, a protein, an antibody, a polymer, a toxin. In some embodiments, the other molecule is an FN3 domain that binds to a molecule other than human albumin. In some embodiments, the other FN3 domain binds to CD71. In some embodiments, the described FN3 domain is linked directly to another molecule. In some embodiments, the described FN3 domain is linked to another molecule via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises the sequence of (GS) ₂ (SEQ ID NO:71), (GGGS) ₂ (SEQ ID NO:72), (GGGS)5 (SEQ ID NO:73), (AP) ₂ (SEQ ID NO:74), (AP) ₅ (SEQ ID NO:75), (AP) ₁₀ (SEQ ID NO: ₇₆ ), (AP) ₂₀ (SEQ ID NO:77), and A(EAAAK) _5AAA (SEQ ID NO:78).

In certain embodiments, the present application provides an FN3-Y fusion represented by the formula: FN3-X-Y or Y-X FN3, where FN3 is an FN3 domain described herein (including any N-terminal and/or C-terminal extensions), X is a polypeptide linker (suitable linkers include, for example, any one of SEQ ID NOs:71-78), and Y is a therapeutic agent moiety described herein.

In certain embodiments, the application provides FN3-Y fusions represented by the formula: FN3-Xi-Cys-X ₂ -Y or Y-Xi-Cys-X ₂ -FN3, where FN3 is an FN3 domain as described herein (including any N-terminal and/or C-terminal extension), Xi is any polypeptide linker (suitable linkers include, for example, any one of SEQ ID NOs:71-78), Cys is a cysteine residue, X ₂ is a chemically derived spacer, and Y is a therapeutic drug moiety as described herein. In an exemplary embodiment, the chemically derived spacer comprises a maleimide moiety that can be used to conjugate a therapeutic drug moiety to the C-terminal Cys of the described FN3 domain or to conjugate a described FN3 domain to the C-terminal Cys of a therapeutic drug moiety by Michael addition, as further described herein. In other aspects, the described FN3 domains may be linked to two or more therapeutic drug moieties. For example, the two moieties can be attached to the described FN3 domain in various configurations from the N-terminus to the C-terminus of the fusion sequence, e.g., X-Y-FN3, X-FN3-Y, or FN3-X-Y, where X and Y represent two different therapeutic therapeutic moieties, selected from any of the moieties disclosed herein.

In certain embodiments, the bispecific FN3 molecule comprises a first FN3 domain and a second FN3 domain, the first FN3 domain comprises a serum albumin-binding FN3 domain described herein, and the second FN3 domain binds to a target protein other than human serum albumin.

Deimmunization of binding polypeptides The amino acid sequence of serum albumin binders and their fusions can be altered to remove one or more B-cell or T-cell epitopes. Proteins comprising the FN3 domain fusions described herein can be deimmunized to render them non-immunogenic or less immunogenic to a given species. Deimmunization can be achieved by structural changes to the protein. Any deimmunization technique known to those skilled in the art can be used, see, for example, WO 00/34317, the disclosure of which is incorporated herein in its entirety.

In one embodiment, the sequences of serum albumin binders and their fusions can be analyzed for the presence of MHC class II binding motifs. Comparisons can be made to databases of MHC binding motifs, for example by searching the "motif" database on the World Wide Web at sitewehil.wehi.edu.au. Alternatively, MHC class II binding peptides can be identified using computational threading methods such as those devised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)), whereby consecutive overlapping peptides from a polypeptide are tested for their binding energy to MHC class II proteins. Computational binding prediction algorithms include iTope™, Tepitope, SYFPEITHI, EpiMatrix (EpiVax), and MHCpred. To aid in the identification of MHC class II binding peptides, one can search for relevant sequence features associated with well-presented peptides, such as amphipathic and Rothbard motifs, as well as cleavage sites for cathepsin B and other processing enzymes.

After identifying potential (e.g. human) T-cell epitopes, these epitopes are eliminated by alteration of one or more amino acids required to eliminate the T-cell epitope. Usually, this involves alteration of one or more amino acids in the T-cell epitope itself. This may involve altering amino acids adjacent to the epitope in terms of the primary structure of the protein, or amino acids that are not adjacent in the primary structure but are adjacent in the secondary structure of the molecule. The usual alterations envisaged are amino acid substitutions, although in certain circumstances addition or deletion of amino acids may be appropriate. All alterations can be achieved by recombinant DNA techniques so that the final molecule can be prepared by expression from a recombinant host, for example by well-established methods, although the use of protein chemistry, or other means of molecular modification, can also be used.

After removal of identified T cell epitopes, the deimmunized sequence can be analyzed again to ensure that no new T cell epitopes have arisen, and if so, these epitopes can be deleted.

It is not necessary to remove all T cell epitopes identified by computation. Those skilled in the art will understand the importance of the "strength" or rather potential immunogenicity of a particular epitope. Various computational methods generate a score of potential epitopes. Those skilled in the art will recognize that only high scoring epitopes may need to be removed. Those skilled in the art will also recognize that there is a balance between removing potential epitopes and maintaining the binding affinity or other biological activity of the protein. Thus, one strategy is to sequentially introduce substitutions into the described FN3 domain or FN3 domain fusion protein and then test for target binding or other biological activity and immunogenicity.

Additional Modifications In certain embodiments, serum albumin binders and their fusions may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. As a result, modified serum albumin binders and their fusions may contain non-amino acid elements such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to a polypeptide. Sialic acid moieties improve solubility and serum half-life while reducing the immunogenic potential of the protein. See, for example, Raju et al., Biochemistry, 2001 Jul. 31; 40(30):8868-76. The effect of such non-amino acid elements on the functionality of a serum albumin binder or its fusions is tested for their ability to bind to a particular serum albumin (e.g., HSA or RhSA) and/or the functional role conferred by a particular non-FN3 moiety in the context of the fusion (e.g., the effect of FGF21 on glucose uptake).

Vector & Polynucleotide Embodiments The present disclosure also includes nucleic acid sequences that code for any of the proteins described herein. As will be understood by those skilled in the art, almost all amino acids can be represented by more than one triplet codon in the coding nucleotide sequence due to the degeneracy of the third base. Furthermore, minor base pair changes may result in conservative substitutions of the encoded amino acid sequence, but are not expected to substantially alter the biological activity of the gene product. Thus, the nucleic acid sequences that code for the proteins described herein can still code for the respective gene products even if the sequence is slightly modified.

Nucleic acids encoding any of the various proteins or polypeptides disclosed herein can be chemically synthesized. Codon usage may be selected to improve expression in a cell. Such codon usage will depend on the type of cell selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See, for example, Mayfield et al., Proc Natl Acad Sci USA. 2003, 100(2):438-42; Sinclair et al., Protein Expr Purif. 2002(1):96-105; Connell ND. Curr Opin Biotechnol. 2001(5):446-9; Makrides et al., Microbiol Rev. 1996, 60(3):512-38; and Sharp et al., Yeast. 1991, 7(7):657-78.

General techniques for nucleic acid manipulation are within the purview of those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2nd Edition, 1989, which are incorporated herein by reference, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic revisions. The DNA encoding the protein is operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. Additionally incorporated are the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. Suitable regulatory elements are well known in the art.

The proteins and fusion proteins described herein can be produced as fusion proteins with a heterologous polypeptide, preferably a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In the case of prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. In the case of yeast secretion, the native signal sequence can be, for example, the yeast invertase leader, factor leaders (including the Saccharomyces and Kluyveromyces alpha factor leaders), or the acid phosphatase leader, C. The C. albicans glucoamylase leader, or the signal described in WO 90/13646, can be substituted. For mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, e.g., the herpes simplex gD signal, are available. The DNA of such precursor regions is ligated in frame to the DNA encoding the protein.

Expression vectors used in eukaryotic host cells (e.g., nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) will also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5', and sometimes 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the multivalent antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

The recombinant DNA can also include any type of protein tag sequence that may be useful for purifying the protein. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual (Elsevier, New York, 1985), the relevant disclosures of which are incorporated herein by reference.

As will be apparent to one of skill in the art, the expression construct is introduced into the host cell using a method appropriate to the host cell. A variety of methods are known in the art for introducing nucleic acids into a host cell, including, but not limited to, electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (where the vector is an infectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells. Suitable bacteria include gram-negative or gram-positive bacteria, e.g., E. coli or Bacillus spp. Yeast, preferably yeasts of the Saccharomyces species such as S. cerevisiae, can also be used for the production of polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers (Bio/Technology, 6:47, 1988). It may also be desirable to produce proteins in vertebrate cells for glycosylation, etc., and growth of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. For many applications, the small size of the protein multimers described herein will make E. coli the preferred method for expression.

Protein Production Host cells are transformed with the expression or cloning vectors described herein for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the proteins of the present invention are cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, the methods described in Ham et al., Meth. S. A. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; International Publication No. WO 90/03430; International Publication No. WO 87/00195; or U.S. Patent Reissue No. 30,985 can be used as the culture medium for host cells. Any of these media can be supplemented with hormones and/or other growth factors (such as insulin, transferrin, epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleotides (such as adenosine, thymidine), antibiotics (such as GENTAMYCIN (trademark) drug), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy source, if necessary. Other necessary supplements can also be included at appropriate concentrations that will be known to those skilled in the art. Culture conditions such as temperature, pH, etc. will be those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

The proteins disclosed herein can also be produced using cellular translation systems. For such purposes, the nucleic acid encoding the protein must be modified to allow in vitro transcription to generate mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized. Exemplary eukaryotic cell-free translation systems include, for example, mammalian or yeast cell-free translation systems, and exemplary prokaryotic cell-free translation systems include, for example, bacterial cell-free translation systems.

The proteins disclosed herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd Ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the proteins can also be produced by chemical synthesis.

The proteins disclosed herein can be purified by protein isolation/purification methods commonly known in the art of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the protein can be exchanged and/or concentrated into a different buffer by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

A purified protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of the purity, the protein is sufficiently pure for use as a pharmaceutical.

Imaging, diagnosis and other uses The FN3 domain fusions provided herein can be used to treat various diseases and disorders based on the identity of the heterologous molecule fused to the described FN3 domain.The use of FN3 domain fusions can be determined by those skilled in the art based on the knowledge of the art and the information provided herein.The use of various FN3 domain fusion proteins is described in detail herein.The FN3 domain fusions can be administered to any mammalian subject or patient, including both human and non-human organisms.

The serum albumin binders and fusion molecules described herein are detectably labeled and used to contact cells expressing the protein bound by the fusion molecule, for example, for imaging or diagnostic applications. Any method known in the art can be employed to conjugate the protein to a detectable moiety, including those described by Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

In certain embodiments, the serum albumin binders and fusion molecules described herein are further conjugated to a detectable label (e.g., the label is a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The label may be a radioactive heavy metal such as an iron chelate, a radioactive gadolinium or manganese chelate, a positron emitter of oxygen, nitrogen, iron, ^carbon , or gallium, ^43K , ^52Fe , ^57Co , 67Cu, ^67Ga , ^68Ga , ^123I , ^125I , ^13T , ^132I , or ^99Tc . The serum albumin binders or fusion molecules conjugated to such moieties can be used as imaging agents and are administered in an amount effective for diagnostic use in a mammal, such as a human, and the localization and accumulation of the imaging agent is then detected. The localization and accumulation of the contrast agent can be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography or positron emission tomography.As those skilled in the art will appreciate, the amount of radioisotope administered depends on the radioisotope.Those skilled in the art can easily formulate the amount of contrast agent administered based on the specific activity and energy of a given radionuclide used as active moiety.

Serum albumin binders and fusion molecules are also useful as affinity purification agents. In this process, the protein is immobilized on a suitable support, such as Sephadex resin or filter paper, using methods known in the art. The protein can be used in known assay methods such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)). Typical Examples

Therapeutic Formulations and Modes of Administration The present invention provides methods for administering a therapeutic moiety fused to a described FN3 domain, where the half-life of the therapeutic moiety is extended when fused to a described FN3 domain. Techniques and dosages for administration of the fusion constructs will vary depending on the type of therapeutic moiety fused to a described FN3 domain and the particular condition being treated, but can be readily determined by one of skill in the art. Regulatory agencies generally require that protein reagents used as therapeutics be formulated to contain acceptably low levels of pyrogens. Thus, therapeutic formulations will generally be distinguished from other formulations in that they are substantially pyrogen-free, or at least contain no more than an acceptable level of pyrogens as determined by the appropriate regulatory agency (e.g., FDA). In certain embodiments, pharmaceutical formulations of the described FN3 domains and their fusion molecules include, for example, 1-20 mM succinic acid, 2-10% sorbitol, and 1-10% glycine at pH 4.0-7.0. In an exemplary embodiment, pharmaceutical formulations of the described FN3 domains and their fusion molecules include, for example, 10 mM succinic acid, 8% sorbitol, and 5% glycine at pH 6.0.

In some embodiments, the described FN3 domains and fusions thereof are pharma- ceutically acceptable to mammals, particularly humans. A "pharma-ceutically acceptable" polypeptide refers to a polypeptide that may be administered to an animal without significant adverse medical effects. Examples of pharma-ceutically acceptable FN3 domains and fusions thereof disclosed herein include FN3 domains lacking the integrin binding domain (RGD) and compositions that are essentially endotoxin-free or have very low levels of endotoxin.

Therapeutic compositions can be administered in unit dosage form with a pharma- ceutically acceptable diluent, carrier, or excipient. By way of non-limiting example, administration can be parenteral (e.g., intravenous, subcutaneous), oral, or topical. The composition can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; a liquid for intravenous, subcutaneous, or parenteral administration; or a gel, lotion, ointment, cream, or polymeric or other sustained release vehicle for topical administration.

Methods well known in the art for preparing formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., A.R. Gennaro A.R., ed., 2000, Lippincott Williams & Wilkins, Philadelphia, PA). Formulations for parenteral administration may contain, for example, excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compound. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation will vary depending on a number of factors, including the dose of drug administered and the route of administration.

The polypeptide may optionally be administered as a pharma- ceutically acceptable salt, such as a non-toxic acid addition salt or metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid, carboxymethylcellulose; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Metal complexes include zinc, iron, and the like. In one example, the polypeptide is formulated in the presence of sodium acetate to enhance thermal stability.

Formulations for oral use include tablets containing the active ingredient in a mixture with non-toxic pharma- ceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium.

A therapeutically effective dose refers to the dose at which it is administered that produces a therapeutic effect. The exact dose depends on the disorder being treated and will be ascertained by one of skill in the art using known techniques. In general, the FN3 domain fusion is administered at about 0.01 μg/kg to about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per day, and most preferably 0.1 mg/kg to about 20 mg/kg per day. The polypeptide may be given daily (e.g., once, twice, three times, or four times per day) or less frequently (e.g., once every other day, once or twice a week, or monthly). Additionally, as known in the art, adjustments for age and weight, general health, sex, diet, time of administration, drug interactions, and severity of disease may be necessary, but will be ascertained by routine experimentation by one of skill in the art.

Kits for detecting human serum albumin Provided herein are kits for detecting human serum albumin in a biological sample, comprising one or more of the serum albumin-binding FN3 domains described herein and instructions for use of the kit.

The serum albumin-binding FN3 domains provided may be in solution; lyophilized; immobilized on a substrate, carrier, or plate; or detectably labeled.

The described kits may also include additional components useful for carrying out the methods described herein. By way of example, the kits may include means for obtaining a sample from a subject, a control or reference sample, e.g., a sample from a subject with a slowly progressing cancer and/or a subject without cancer, one or more sample compartments, and/or educational materials describing the performance of the methods of the invention and tissue-specific controls or standards.

The means for determining the level of serum albumin can further include, for example, a buffer or other reagent for use in an assay to determine the level of serum albumin. The instructions can be, for example, printed instructions for performing an assay and/or instructions for assessing the level of serum albumin.

The described kits may also include means for isolating a sample from a subject. These means may include one or more items of equipment or reagents that can be used to obtain fluid or tissue from a subject. The means for obtaining a sample from a subject may also include means for isolating a blood component, such as serum, from a blood sample. Preferably, the kit is designed for use with human subjects.

Embodiments described herein are directed to a method of detecting the presence of human serum albumin in a biological sample, comprising contacting the biological sample with an albumin-binding FN3 domain described herein and assessing binding of the biological sample to the protein.

The embodiments described herein are directed to a method of extending the half-life of a target molecule in a human subject, comprising binding a serum albumin-binding FN3 domain described herein to the target molecule, thereby extending the half-life of the target molecule in a human subject. In some embodiments, the method further comprises administering the conjugated molecule to a human. In some embodiments, the target molecule is a drug, an antibody, an FN3 domain that binds to a molecule other than human serum albumin, or a toxin. In some embodiments, the conjugation is a peptide linker. In some embodiments, the peptide linker comprises the sequence of (GS) ₂ (SEQ ID NO:71), (GGGS)2 (SEQ ID NO:72), (GGGS) ₅ (SEQ ID NO:73), (AP) ₂ (SEQ ID NO:74), (AP) ₅ (SEQ ID NO:75), (AP) ₁₀ (SEQ ID NO:76), (AP) ₂₀ (SEQ ID NO:77) _, and A(EAAAK) _5AAA (SEQ ID NO:78).

The following examples are provided to supplement the previous disclosure and provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter described. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be apparent to those skilled in the art and are within the true scope of the invention and can be made without departing from it.

Construction of TENCON Libraries Using Randomized Loops Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain designed from the consensus sequence of 15 FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Patent No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops connecting seven beta-strands. These loops, or selected residues within each loop, may be randomized to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

Tencon:

Using the Tencon scaffold and different design strategies, different libraries were generated. In general, libraries TCL1 and TCL2 generated superior binders. The construction of the TCL1 and TCL2 libraries is described in detail in WO2014081944.

Construction of the TCL1 Library A library designed to randomize only the FG loop, TCL1, of Tencon (SEQ ID NO:1) was constructed for use in a cis-display system (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012). In this system, double-stranded DNA is generated that incorporates the sequences of the Tac promoter, the Tencon library coding sequence, the RepA coding sequence, the cis element, and the ori element. Upon expression in an in vitro transcription/translation system, a complex is generated in which the Tencon-RepA fusion protein and the DNA encoding it are bound in cis. Complexes that bind to the target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

Construction of the TCL1 library for use in Cis display was accomplished by successive rounds of PCR to generate a final linear double-stranded DNA molecule divided into two halves; the 5' fragment contains the promoter and Tencon sequences, and the 3' fragment contains the repA gene and the cis and ori elements. These two halves are combined by restriction digestion to generate the entire construct. The TCL1 library was designed to incorporate random amino acids only into the Tencon FG loop KGGHRSN (SEQ ID NO:32). NNS codons were used in the construction of this library, resulting in all 20 amino acids and one stop codon being potentially incorporated into the FG loop. The TCL1 library contains six separate sub-libraries, each with a different randomized FG loop length of 7 to 12 residues, to further increase diversity.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_７は、任意のアミノ酸であり；そして
Ｘ_８、Ｘ_９、Ｘ_１０、Ｘ_１１およびＸ_１２は、任意のアミノ酸であるか、または削除されている TCL1 library (SEQ ID NO:2)

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X7 are any amino acid; and _X8 , _X9 , _X10 , _X11 and _X12 are any amino acid or are deleted.

Construction of the TCL2 Library A TCL2 library was constructed in which both the BC and FG loops of Tencon were randomized and the distribution of amino acids at each position was tightly controlled. Table 2 shows the amino acid distribution at the desired loop positions in the TCL2 library. The designed amino acid distribution had two objectives. First, the library had a bias towards residues predicted to be structurally important for Tencon folding and stability based on analysis of the crystal structure of Tencon and/or from homology modeling. For example, position 29 was fixed to only a subset of hydrophobic amino acids because this residue is buried in the hydrophobic core of the Tencon fold. The second layer of design involved biasing the amino acid distribution towards residues preferentially found in the heavy chain HCDR3 of antibodies to efficiently generate high affinity binders (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). To this end, the "Designed Distribution" in Table 2 refers to the following distribution: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution does not include methionine, cysteine, or a stop codon.

式中、
Ｘ_１は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_２は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_３は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_４は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_５は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_６は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_７は、Ｐｈｅ、Ｉｌｅ、Ｌｅｕ、ＶａｌまたはＴｙｒであり；
Ｘ_８は、Ａｓｐ、ＧｌｕまたはＴｈｒであり；
Ｘ_９は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１０は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１１は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１２は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１３は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１４は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；そして
Ｘ_１５は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌである。 TCL2 library (SEQ ID NO:3)

During the ceremony,
_X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X7 is Phe, Ile, Leu, Val or Tyr;
_X8 is Asp, Glu or Thr;
_X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and _X15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

These libraries were then improved in a variety of ways, including building libraries on a stabilized Tencon framework (US Pat. No. 8,569,227) incorporating substitutions E11R/L17A/N46V/E86I (Tencon27; SEQ ID NO: 4) compared to wild-type Tencon, and altering the randomized positions in the BC and FG loops. Tencon27 is described in WO2013049275. From this, new libraries were generated that were designed to randomize only the FG loop of Tencon (library TCL9) or a combination of the BC and FG loops (library TCL7). These libraries were constructed for use in cis-display systems (Odegrip et al., Proc Natl Acad Sci USA 101:2806-2810, 2004). Details of this design are provided below:

Stabilized Tencon (Tencon27) (SEQ ID NO:4)

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２、Ｘ_１３、Ｘ_１４、Ｘ_１５およびＸ_１６は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、ＷまたはＹ；であり、そして
Ｘ_７、Ｘ_８、Ｘ_９、Ｘ_１７、Ｘ_１８およびＸ_１９は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙであるか、または削除されている。 TCL7 (randomized FG and BC loops) (SEQ ID NO:5)

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 , _X13 , X14, _X15 and _X16 are A, D, E, F, G, H, I, K, L, N, P, Q, _R , S, T, V, W or Y; and _X7 , _X8 , _X9 , _X17 , _X18 and _X19 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６およびＸ_７は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、ＷまたはＹであり；そして
Ｘ_８、Ｘ_９、Ｘ_１０、Ｘ_１１およびＸ_１２は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙであるか、または削除されている。 TCL9 (randomized FG loop) (SEQ ID NO:6)

_X1 , _X2 , _X3 , _X4 , _X5 , _X6 and _X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and _X8 , _X9 , _X10 , _X11 and _X12 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

For library construction, DNA fragments encoding randomized BC loops (length 6-9 positions) or FG loops (length 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) to control the amino acid distribution of the library and eliminate stop codons. Two different sets of DNA molecules randomizing either the BC or FG loop were synthesized separately and later combined using PCR to generate the complete library product.

Construction of FG loop library (TCL9)
A set of synthetic DNA molecules was generated consisting of the complete gene sequence of Tencon, except for the 5'Tac promoter followed by randomized codons in the FG loop (SEQ ID NO:26-31). The randomization of the FG loop encoded all amino acids except cysteine and methionine in equal proportions. The lengths of the diversified parts were such that they encoded 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sublibraries of each length variation were synthesized separately at a 2 μg scale and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO:9) and Sloning-Rev (SEQ ID NO:10).

The 3' fragment of the library is a constant DNA sequence containing elements for presentation, including a PspOMI restriction site, the coding region of the repA gene, and cis and ori elements. To amplify this fragment, a PCR reaction was performed using M13 Forward and M13 Reverse primers with the plasmid (pCR4Blunt) (Invitrogen) as a template. The resulting PCR product was digested overnight with PspOMI and gel purified. To ligate the 5' portion of the library DNA to the 3' DNA containing the repA gene, 2 pmol (approximately 540 ng to 560 ng) of 5' DNA was ligated to equimolar (approximately 1.25 μg) of 3'repA DNA in the presence of NotI and PspOMI enzymes and T4 ligase overnight at 37°C. The ligated library products were amplified using 12 cycles of PCR with oligo POP2250 (SEQ ID NO:11) and DigLigRev (SEQ ID NO:12). For each sublibrary, DNA from the 12 PCR reactions was combined and purified on a Qiagen spin column. Yields for each TCL9 sublibrary ranged from 32-34 μg.

Construction of the FG/BC Loop Library (TCL7) The TCL7 library provides a library with randomized Tencon BC and FG loops. In this library, BC loops of 6-9 amino acids in length were combinatorially mixed with randomized FG loops of 7-12 amino acids in length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NOs: 13-16) were made containing the Tencon gene encoding the N-terminal portion of the protein up to and including residue VX, such that the BC loop was replaced with 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized before the discovery of the L17A, N46V, and E83I mutations (CEN5243), which were introduced in the molecular biology steps described below. To combine this fragment with the fragment encoding the randomized FG loop, the following steps were performed.

First, a DNA fragment encoding the Tac promoter and the 5' sequence of Tencon up to the nucleotide encoding amino acid A17 (130mer-L17A, SEQ ID NO:17) was generated by PCR using oligos POP2222ext (SEQ ID NO:18) and LS1114 (SEQ ID NO:19). This was done to include the L17A mutation in the library (CEN5243). Next, a DNA fragment encoding Tencon residues R18-V75, including the randomized BC loop, was amplified by PCR using BC6, BC7, BC8, or BC9 as templates and oligos LS1115 (SEQ ID NO:20) and LS1117 (SEQ ID NO:21). This PCR step introduced a BsaI site at the 3' end. These DNA fragments were then joined by overlap PCR using oligos POP2222ext and LS1117 as primers. The resulting 240 bp PCR products were pooled and purified using the Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified.

To incorporate a BsaI restriction site and the N46V and E86I mutations (CEN5243), the fragment encoding the FG loop was amplified by PCR with oligonucleotides SDG10 (SEQ ID NO:22) and SDG24 (SEQ ID NO:23) using FG7 (SEQ ID NO:31), FG8 (SEQ ID NO:30), FG9 (SEQ ID NO:29), FG10 (SEQ ID NO:28), FG11 (SEQ ID NO:27), and FG12 (SEQ ID NO:26) as templates.

The digested BC and FG fragments were ligated together in one step using a three-way ligation. Four ligation reactions were set up with 16 possible combinations, with each ligation combining two BC and two FG loop lengths. Each ligation contained approximately 300 ng of total BC fragments and 300 ng of FG fragments. These four ligation pools were then amplified by PCR with oligos POP2222 (SEQ ID NO:24) and SDG28 (SEQ ID NO:25). 7.5 μg of each reaction product was then digested with Not1 and cleaned up with a Qiagen PCR purification column. 5.2 μg of this DNA was ligated to an equimolar amount of RepA DNA fragment (approximately 14 μg) digested with PspOMI, and the product was amplified by PCR using oligo POP2222.

Generation of TENCON Libraries with Alternative Binding Surfaces The choice of residues to be randomized in a particular library design determines the overall shape of the interaction surface generated. X-ray crystallography of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a highly curved interface that fits into the active site of MBP (Koide et al., Proc Natl Acad Sci USA 104:6632-6637, 2007). In contrast, an ankyrin repeat scaffold protein selected to bind MBP was found to have a much more planar interaction surface and bind to the outer surface of MBP, away from the active site (Binz et al., Nat Biotechnol 22:575-582, 2004). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may determine which target proteins or specific epitopes on those target proteins can be effectively bound by the scaffold. Published efforts to engineer protein scaffolds containing FN3 domains for protein binding have relied on engineering adjacent loops for target binding, thus generating curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops located on opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by a beta strand that forms the center of the FN3 structure. The image of Tencon can be rotated 90 degrees to visualize the other surface. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta strands, the C and F beta strands, and is referred to here as the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein-scaffold interaction surfaces by randomizing a subset of the residues that form the surface. The beta strands have a repeating structure in which every other residue's side chain is exposed to the surface of the protein. Thus, libraries can be created by randomizing some or all of the surface-exposed residues of the beta strands. Selection of appropriate residues in the beta strands should minimize loss of the inherent stability of the Tencon scaffold and provide a unique scaffold surface for interaction with other proteins.

Library TCL14 (SEQ ID NO:7) was designed in the Tencon27 scaffold (SEQ ID NO:4).

A complete description of the methods used to construct this library is provided in U.S. Patent Application Publication No. 2013/0226834.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２およびＸ_１３は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙ、またはＭである。 TCL14 library (SEQ ID NO:7):

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 and _X13 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or M.

The two beta strands that form the C-CD-F-FG surface of Tencon27 have a total of nine surface-exposed residues that can be randomized: C strand: S30, L32, Q34, Q36; F strand: E66, T68, S70, Y72, and V74, while the CD loop has six potential residues: S38, E39, K40, V41, G42, and E43, and the FG loop has seven potential residues: K75, G76, G77, H78, R79, S80, and N81. Selected residues were chosen for inclusion in the design of TCL14 because the theoretical size of the library would be large if all 22 residues were randomized.

Thirteen positions in Tencon were selected for randomization: L32, Q34, and Q36 in the C strand, S38, E39, K40, and V41 in the CD loop, T68, S70, and Y72 in the F strand, and H78, R79, and N81 in the FG loop. In the C and F strands, S30 and E66 were not randomized because they are just beyond the CD and FG loops and are not apparently part of the C-CD-F-FG surface. In the CD loop, G42 and E43 were not randomized because glycine provides flexibility and can be useful in the loop region, and E43 is at the interface of the surface. In the FG loop, K75, G76, G77, and S80 were excluded. Glycine was excluded for the reasons mentioned above, but careful examination of the crystal structure revealed that S80 forms important contacts with the core and helps to form a stable FG loop. K75 faces away from the surface of the C-CD-F-FG surface and was a less attractive candidate for randomization. Although the above residues were not randomized in the original TCL14 design, they can be included in subsequent library designs to provide additional diversity for de novo selection or, for example, affinity maturation libraries for hits specific to the selected TCL14 target.

Following the generation of TCL14, three additional Tencon libraries of similar design were generated. These two libraries, TCL19, TCL21, and TCL23, are randomized at the same positions as TCL14 (see above), but the distribution of amino acids occurring at these positions is altered (Table 3). TCL19 and TCL21 are designed to have the 18 natural amino acids evenly distributed at all positions (5.55% each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loop of a functional antibody, as described in Table 2 (Birtalan et al., J Mol Biol 377:1518-1528, 2008). As with the TCL21 library, cysteine and methionine were excluded.

To expand the potential target binding surface of the other libraries, a third additional library was constructed. In this library, TCL24, four additional Tencon positions were randomized compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D strand, and S84 and I86 from the G strand. Positions 46, 48, 84, and 86 were specifically selected because the side chains of these residues are surface exposed from beta strands D and G and are structurally adjacent to the randomized portions of the C and F strands, increasing the surface area accessible for binding to the target protein. The amino acid distribution used at each position in TCL24 is the same as that described for TCL19 and TCL21 in Table 3.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２およびＸ_１３は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、ＶまたはＷである。 TCL24 library (SEQ ID NO:8)

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 and _X13 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W.

Generation of TCL21, TCL23, and TCL24 Libraries The TCL21 library was generated using Colibra library technology (Isogenica) to control the amino acid distribution. The TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control the amino acid distribution. Following initial synthesis, each library was amplified using PCR for selection with the CIS display system (Odegrip et al., Proc Natl Acad Sci USA 101:2806-2810, 2004) as described above for the loop library, followed by ligation to the gene for RepA.

Selection library screening of fibronectin type III (FN3) domains that bind human serum albumin Cis display was used to select human serum albumin binding domains from the TCL14, TCL19, TCL21, TCL23, and TCL24 libraries. Recombinant human and cynomolgus serum albumin and human albumin domain II (Albumin Biosciences) were biotinylated using standard methods and used for panning. For in vitro transcription and translation (ITT), 3 μg of library DNA was incubated at 30°C with a total volume of 50 μL of 0.1 mM complete amino acids, 1× S30 premix components, and 15 μL of S30 extract (Promega). After 1 h, 375 μL of blocking solution (0.1% casein (Thermo Fisher, Rockford, IL), 100 mg/ml herring sperm DNA (Promega, Madison, WI), 1 mg/ml heparin (Sigma-Aldrich, St. Louis, MO)) was added and the reaction was incubated on ice for 15 min. For selection, biotinylated antigen was added at concentrations of 400 nM (round 1), 200 nM (rounds 2 and 3) and 100 nM (rounds 4 and 5). Bound library members were recovered using neutravidin magnetic beads (Thermo Fisher, Rockford, IL) (rounds 1, 3, and 5) or streptavidin magnetic beads (Promega, Madison, WI) (rounds 2 and 4), and unbound library members were removed by washing the beads 5-14 times with 500 μL PBST followed by two times with 500 μL PBS. Additional selection rounds were performed to identify FN3 domain molecules with improved affinity. Briefly, the output from round 5 was prepared as above and subjected to additional iterative rounds of selection with the following modifications: incubation with biotinylated antigen was shortened from 1 hour to 15 minutes, bead capture was shortened from 20 minutes to 15 minutes, bt-HSA was reduced to 25 nM (rounds 6 and 7) or 2.5 nM (rounds 8 and 9), and an additional hour of washing was performed in the presence of excess non-biotinylated target protein. The purpose of these modifications was to simultaneously select for binders with potentially faster on-rates and slower off-rates resulting in a substantially lower _KD .

After panning, selected FN3 domains were amplified by PCR using oligos Tcon6 (SEQ ID NO:33) and Tcon5shortE86I (SEQ ID NO:34), subcloned into pET15-LIC by annealing, and transformed into BL21-GOLD(DE3) cells (Agilent, Santa Clara, CA) for soluble expression in E. coli using standard molecular biology techniques. Single clones were picked and grown to saturation in 1 mL LB with ampicillin in 96 deep-well plates at 37°C. The next day, 25 μL was transferred to 1 mL fresh LB-Amp medium in 96 deep-well plates and incubated at 37°C for 2 h. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 30°C for 16 h. Cells were harvested by centrifugation and then lysed with Bugbuster HT (EMD Chemicals, Gibbstown, NJ) supplemented with hen egg white lysozyme (Sigma-Aldrich, St. Louis, MO) at a final concentration of 0.2 mg/mL. Bacterial lysates were cleared by centrifugation and the supernatants were transferred to new 96-deep-well plates and tested for binding to target proteins by ELISA.

Selection of FN3 domains that bind human serum albumin To identify human serum albumin binders, enzyme-linked immunosorbent assays (ELISAs) were performed on individual clones from selected panning outputs. Maxisorp plates (Nunc, Rochester, IL) were coated overnight with 5 μg HSA, cynomolgus SA or 5 μg/ml Fc (Sigma-Aldrich, St. Louis, MO), washed with Tris-buffered saline, pH 7.4, containing 0.05% Tween-20 (TBST), and blocked using Starting Block T20 (Thermo Fisher, Rockford, IL). Clarified bacterial lysates (as above) were applied to the wells of the coated HSA, cSA and Fc plates. Plates were incubated for 1 h, washed with TBST, and bound centirin was detected with anti-V5 tag antibody and POD chemiluminescent substrate (Roche, Indianapolis, IN) using a Molecular Devices M5 plate reader. Hits were defined as human and cynomolgus serum albumin binding signals >10 relative to the Fc binding signal.

One FN3 domain, B7, showed significant binding to human and cynomolgus albumin, as well as to domain II of human albumin, as shown in Table 4 below. Residues important for binding to human SA were further characterized by preparing variants in which each residue in the putative binding site was individually mutated to alanine. Table 5 shows the complete amino acid sequence of the FN3 domains and alanine variants that bind to domain II. SEQ ID NO:51 was compared to the sequence of H9 (SEQ ID NO:70) and found to have a significantly increased half-life in cynomolgus monkeys. This was unexpected.

Small-scale expression and purification identified FN3 domains that bind human serum albumin. Clones of selected FN3 domains were picked and grown to saturation in 1 mL Luria Broth (LB) supplemented with 100 μg/mL ampicillin (LB-Amp medium) at 37°C in 96 deep-well plates. The next day, 25 μL was transferred to 5 mL fresh LB-Amp medium in a 24 deep-well plate and incubated at 37°C for 2 hours. IPTG was added to a final concentration of 1 mM and protein expression was induced at 30°C for 16 hours. Cells were harvested by centrifugation and lysed with Bugbuster HT (EMD Chemicals, Gibbstown, NJ) supplemented with hen egg white lysozyme (Sigma-Aldrich, St. Louis, MO) at a final concentration of 0.2 mg/mL. Bacterial lysates were cleared by centrifugation and the supernatants were transferred to new 96-deep-well plates. His-tagged FN3 domains were purified using 96-well Ni-NTA multitrap plates according to the manufacturer's recommendations (GE Lifesciences, Piscataway, NJ).

Size Exclusion Chromatography Analysis Size exclusion chromatography was used to determine the aggregation state of FN3 domains bound to human serum albumin. An aliquot (10 μL) of each purified FN3 domain was injected onto a Superdex 75 5/150 column (GE Healthcare) in a mobile phase of PBS pH 7.4 at a flow rate of 0.3 mL/min. Elution from the column was monitored by absorbance at 280 nm. FN3 domains that showed high levels of aggregation by SEC were excluded from further analysis.

Characterization of Human Serum Albumin Binding FN3 Domains Immunoprecipitation of Human Serum Albumin-FN3 Domain Complexes To assess binder functionality, selected FN3 domains were tested for their ability to complex with endogenous albumin in normal serum. Methods for determining this are provided in U.S. Patent Application No. 15/611,296, which is incorporated herein by reference. Most sequences, even those with substitutions, were found to bind and pull down albumin. Certain positions may not tolerate alanine substitutions, but other substitutions at these positions can be made without eliminating albumin interactions. The results are shown in Table 6.

The FN3 proteins were also tested to determine their ability to bind to immobilized human or cynomolgus albumin in an ELISA assay. Binding of 667 nM B7 and B7 variants to human or cynomolgus albumin is shown in Table 7. The negative control is wild-type Tencon.

Pharmacokinetics of albumin-binding FN3 domains in cynomolgus monkeys:
In vivo studies were performed with B7 and H9. Either B7 or H9 was administered to cynomolgus monkeys at a dose of 5 mg/kg by intravenous (IV) bolus injection. The method of administration and the method for determining the protein concentration were determined according to the method described in US Patent Application No. 15/611296, which is incorporated herein by reference. Figures 1 and 2 show the pharmacokinetics, including the increased half-life, of B7 or albumin FN3 domain, referred to as ALB40.

Sequence information

SEQ ID NO:1 = original Tencon sequence

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_７は、任意のアミノ酸であり；そして
Ｘ_８、Ｘ_９、Ｘ_１０、Ｘ_１１およびＸ_１２は、任意のアミノ酸であるか、または削除されている。 SEQ ID NO:2 = TCL1 library

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X7 are any amino acid; and _X8 , _X9 , _X10 , _X11 and _X12 are any amino acid or are deleted.

式中、
Ｘ_１は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_２は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_３は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_４は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_５は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_６は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_７は、Ｐｈｅ、Ｉｌｅ、Ｌｅｕ、ＶａｌまたはＴｙｒであり；
Ｘ_８は、Ａｓｐ、ＧｌｕまたはＴｈｒであり；
Ｘ_９は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１０は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１１は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１２は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１３は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；
Ｘ_１４は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌであり；そして
Ｘ_１５は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｎ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌである。 SEQ ID NO:3 = TCL2 library

During the ceremony,
_X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X7 is Phe, Ile, Leu, Val or Tyr;
_X8 is Asp, Glu or Thr;
_X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
_X14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and _X15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

SEQ ID NO:4 = stabilized Tencon (Tencon27)

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２、Ｘ_１３、Ｘ_１４、Ｘ_１５およびＸ_１６は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、ＷまたはＹであり；そして
Ｘ_７、Ｘ_８、Ｘ_９、Ｘ_１７、Ｘ_１８およびＸ_１９は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙであるか、または削除されている。 SEQ ID NO:5 = TCL7 (FG and BC loops)

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 , _X13 , X14, _X15 and _X16 are A, D, E, F, G, H, I, K, L, N, P, Q, _R , S, T, V, W or Y; and _X7 , _X8 , _X9 , _X17 , _X18 and _X19 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６およびＸ_７は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、ＷまたはＹであり；そして
Ｘ_８、Ｘ_９、Ｘ_１０、Ｘ_１１およびＸ_１２は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙであるか、または削除されている。 SEQ ID NO:6 = TCL9 (FG loop)

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 and _X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and _X8 , _X9 , _X10 , _X11 and _X12 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２およびＸ_１３は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、Ｖ、Ｗ、Ｙ、またはＭである。 SEQ ID NO:7 = TCL14 library

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 and _X13 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or M.

式中、
Ｘ_１、Ｘ_２、Ｘ_３、Ｘ_４、Ｘ_５、Ｘ_６、Ｘ_１０、Ｘ_１１、Ｘ_１２、Ｘ_１３は、Ａ、Ｄ、Ｅ、Ｆ、Ｇ、Ｈ、Ｉ、Ｋ、Ｌ、Ｎ、Ｐ、Ｑ、Ｒ、Ｓ、Ｔ、ＶまたはＷである。 SEQ ID NO:8 = TCL24 library

During the ceremony,
_X1 , _X2 , _X3 , _X4 , _X5 , _X6 , _X10 , _X11 , _X12 , and _X13 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W.

SEQ ID NO:9 = Sloning-FOR

SEQ ID NO:10 = Sloning-REV

SEQ ID NO:11 = POP2250

SEQ ID NO:12 = DigLigRev

SEQ ID NO:13=BC9

SEQ ID NO:14=BC8

SEQ ID NO:15=BC7

SEQ ID NO:16=BC6

SEQ ID NO:17 = 130mer-L17A

SEQ ID NO:18 = POP222ext

SEQ ID NO:19=LS1114

SEQ ID NO:20=LS1115

SEQ ID NO:21=LS1117

SEQ ID NO:22 = SDG10

SEQ ID NO:23 = SDG24

SEQ ID NO:24=POP2222

SEQ ID NO:25=SDG28

SEQ ID NO:26=FG12

SEQ ID NO:27=FG11

SEQ ID NO:28=FG10

SEQ ID NO:29=FG9

SEQ ID NO:30=FG8

SEQ ID NO:31 = FG7

SEQ ID NO: 32 Tencon FG loop

SEQ ID NO:33 = Tcon6

SEQ ID NO:34 = Tcon5E86Ishort

SEQ ID NO:35 Original Tencon C strand

SEQ ID NO: 36 C strand of ALB-E05

SEQ ID NO: 37 C strand of ALB-E07

SEQ ID NO: 38 C strand of ALB-H9

SEQ ID NO:39 Original Tencon CD loop

SEQ ID NO: 40 CD loop of ALB-E05

SEQ ID NO: 41 CD loop of ALB-E07

SEQ ID NO: 42 CD loop of ALB-H9

SEQ ID NO: 43 Original Tencon F strand

SEQ ID NO: 44 F strand of ALB-E05

SEQ ID NO: 45 F strand of ALB-E07

SEQ ID NO: 46 F strand of ALB-H9

SEQ ID NO: 47 Original Tencon FG loop

SEQ ID NO: 48 FG loop of ALB-E05

SEQ ID NO: 49 FG loop of ALB-E07

SEQ ID NO: 50 FG loop of ALB-H9

B7 - SEQ ID NO:51

A10V SEQ ID NO:52

A10V, N33A SEQ ID NO:53

A10V, A35S SEQ ID NO:54

A10V, W37A SEQ ID NO:55

A10V, P39A SEQ ID NO:56

A10V, G40A SEQ ID NO:57

A10V, I41A SEQ ID NO:58

A10V, G42A SEQ ID NO:59

A10V, W47A SEQ ID NO: 60

A10V, R49A SEQ ID NO:61

A10V, K69A SEQ ID NO:62

A10V, W71A SEQ ID NO: 63

A10V, H73A SEQ ID NO: 64

A10V, A79S SEQ ID NO: 65

A10V, S80A SEQ ID NO:66

A10V, P82A SEQ ID NO:67

A10V, I85A SEQ ID NO:68

A10V, R87A SEQ ID NO: 69

SEQ ID NO:70 ALB-H9

SEQ ID NO:71 (GS) ₂

SEQ ID NO:72 (GGGS) ₂

SEQ ID NO:73 (GGGGS) ₂

SEQ ID NO:74 (AP) ₂

SEQ ID NO: 75 (AP) ₅

SEQ ID NO: 76 (AP) ₁₀

SEQ ID NO:77 (AP) ₂₀

SEQ ID NO:78 A(EAAAK) ₅ AAA

SEQ ID NO: 79 Human serum albumin

SEQ ID NO: 80 Cynomolgus serum albumin

SEQ ID NO: 81 Tenascin C (TN3)

SEQ ID NO:82 Fibcon

SEQ ID NO:83 FN10

Claims (30)

An FN3 domain protein that binds to human serum albumin, the FN3 domain protein comprising the amino acid sequence of SEQ ID NO: 51, 52, 54, 56, 57, 58, 59, 61, 62, 66, 67, or 69, or an FN3 domain protein that binds to human serum albumin, the FN3 domain protein comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51, 52, 54, 56, 57, 58, 59, 61, 62, 66, 67, or 69. The FN3 domain protein of claim 1, comprising a cysteine residue at at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, or 88 of SEQ ID NO:1. The FN3 domain protein of claim 1, further comprising a cysteine at the N-terminus and/or C-terminus of the FN3 domain protein. The FN3 domain protein of claim 1, comprising a substitution at a position corresponding to position 10 of SEQ ID NO:51. The FN3 domain protein of claim 1, wherein the FN3 domain protein comprises substitutions of A10V, N33A, A35S, W37A, P39A, G40A, I41A, G42A, W47A, R49A, K69A, W71A, H73A, A79S, S80A, P82A, I85A, or R87A, and the positions correspond to those in SEQ ID NO:51. The FN3 domain protein of claim 1, comprising a substitution at a position corresponding to positions 10, 33, 25, 37, 39, 40, 41, 42, 47, 49, 69, 71, 73, 79, 80, 82, 85, or 87 of SEQ ID NO:51. The FN3 domain protein of claim 4, wherein the substitution is A10V, A10G, A10L, A10I, A10T, or A10S. The FN3 domain protein according to claim 1, wherein the FN3 domain protein comprises an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:51 or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions when compared to the amino acid sequence of SEQ ID NO:51. The FN3 domain protein according to any one of claims 1 to 8, which is linked to another molecule. The FN3 domain protein of claim 9, wherein the other molecule is a drug or therapeutic agent, a protein, an antibody, a polymer, or a toxin. The FN3 domain protein of claim 9, wherein the other molecule is an FN3 domain that binds to a molecule other than human albumin. The FN3 domain protein of claim 11, wherein the FN3 domain binds to CD71. The FN3 domain protein according to any one of claims 9 to 12, which is directly linked to another molecule. The FN3 domain protein according to any one of claims 9 to 12, which is linked to another molecule via a linker. The FN3 domain protein according to claim 14, wherein the linker is a peptide linker. The FN3 domain protein of claim 15, wherein the peptide linker _comprises the sequence (GS) ₂ (SEQ ID NO:71), (GGGS) ₂ (SEQ ID NO:72), (GGGGS) ₅ (SEQ ID NO:73), (AP) ₂ (SEQ ID NO:74), (AP) ₅ (SEQ ID NO:75), (AP) ₁₀ (SEQ ID NO:76), (AP)20 (SEQ ID NO:77) or A(EAAAK) _5AAA (SEQ ID NO:78). An isolated polynucleotide encoding an FN3 domain protein according to any one of claims 1 to 16 . A vector comprising the polynucleotide of claim 17 . 20. An isolated host cell comprising the vector of claim 18 . 20. A method for producing an FN3 domain protein, comprising culturing the isolated host cell of claim 19 under conditions such that the FN3 domain protein is expressed. 21. The method of claim 20 , further comprising purifying the FN3 domain protein. A pharmaceutical composition comprising the FN3 domain protein according to any one of claims 1 to 16 and a pharma- ceutically acceptable carrier. A method for detecting the presence of human serum albumin in a biological sample, the method comprising contacting the biological sample with an FN3 domain protein according to any one of claims 1 to 16 , and assessing binding of the biological sample to the FN3 domain protein. A kit comprising the FN3 domain protein according to any one of claims 1 to 16 . A bispecific FN3 molecule comprising a first FN3 domain and a second FN3 domain, wherein the first FN3 domain comprises an FN3 domain protein according to any one of claims 1 to 16 , and the second FN3 domain binds to a target protein other than human serum albumin. 26. A pharmaceutical composition comprising the bispecific molecule of claim 25 . A method for extending the half-life of a target molecule in a human subject, comprising conjugating an FN3 domain protein according to any one of claims 1 to 16 to the target molecule, thereby extending the half-life of the target molecule in the human subject. 28. The method of claim 27 , wherein the target molecule is a drug, an antibody, an FN3 domain that binds to a molecule other than human serum albumin, or a toxin. 29. The method of claim 27 or 28 , wherein the conjugation is a peptide linker. 30. The method of _claim 29, wherein the peptide linker comprises the sequence (GS) ₂ (SEQ ID NO:71), (GGGS) ₂ (SEQ ID NO:72), (GGGGS) ₅ (SEQ ID NO:73), (AP) ₂ (SEQ ID NO:74), (AP) ₅ (SEQ ID NO:75), (AP) ₁₀ (SEQ ID NO:76), (AP)20 (SEQ ID NO:77) or A(EAAAK) _5AAA (SEQ ID NO: 78 ).

Images